Endocytosis and cell signaling are important for viability of eukaryotes. In Trypanosoma brucei, little is known about cell signaling because the major receptors studied in model eukaryotes are unrecognizable in the parasite genome. T. brucei has a glycosylphosphatidylinositol-phospholipase C (GPI-PLC) whose biological function(s) of GPI-PLC have been elusive. We found recently that GPI-PLC stimulates endocytosis of transferrin up to 500%, and that enzyme activity is needed for this physiological effect. Therefore, we hypothesized that a cleavage product of GPI-PLC would be sufficient to stimulate endocytosis. Consistent with this concept, exogenous diacylglycerol (DAG) promoted endocytosis in the parasite. Thus, T. brucei has receptors for DAG that regulate endocytosis. Our long-term goals are to delineate the components and evaluate the physiological significance of DAG signaling in T. brucei. Towards these goals, we describe approaches for identification of DAG-binding proteins (TbDAGBPs). In Specific Aim 1, we will (i) clone and characterize TbDAGBPs predicted from bioinformatic analysis;(ii) use conventional chromatography to purify TbDAGBPs;and (iii) screen a cDNA expression library for TbDAGBPs. In Specific Aim 2, effectors that link DAG signaling to the endocytic system will be identified, by knocking down expression of TbDAGBPs, and testing whether the TbDAGBP-deficient T. brucei lose ability to stimulate endocytosis in response to DAG. These studies will lead to discovery of novel DAG binding proteins because the C1-domain used in vertebrates for recognition of DAG is not detectable in the genome of T. brucei. Trypanosomes cause diseases that affect millions of people world-wide. Work described in this proposal may lead to discovery of new signaling pathways that may be targeted for treatment of human African trypanosomiasis.